The DNA damage checkpoints are cellular surveillance mechanisms that allow the cell to monitor the integrity of the genome and to signal the presence of DNA damage to the cell. Checkpoint pathways are frequently disrupted In tumor cells, demonstrating the importance of this response to the maintenance of the genome. Several lines of evidence suggest that inhibitors of the DNA damage checkpoint can be more effective at sensitizing cancer cells to DNA damaging agents than normal cells. Therefore, inhibitors of the DNA damage checkpoint could be clinically useful anti-cancer agents, and preclinical studies support this hypothesis. The broad long-term objectives of this work are to identify and validate new targets for cancer therapy. Studies suggest that ATR, a protein kinase related to members of the lipid kinase family, is a central component of the DNA damage checkpoint in mammalian cells. Inhibition of ATR through expression of a dominant-negative form of the protein selectively sensitizes cells with abnormal Gl/S regulation to DNA damaging agents. Moreover caffeine, an inhibitor of ATR and the related kinase ATM, selectively sensitizes p53-deficient cells to several DNA damaging agents. These data suggest ATR may be a useful target for cancer treatment. The project proposed in this application is to develop two assays to screen for small molecule inhibitors of ATR and other checkpoint proteins. ATR is a DNA binding protein and studies suggest that the interaction of ATR with DNA is mediated by another protein(s). One assay will be designed to screen for small molecule that inhibit this interaction in vitro. Because multiple proteins are involved in this interaction, inhibitors of ATR and its associated DNA binding activity can be sought simultaneously. In the second assay, compounds that bind directly to ATR will be sought using a small-molecule microarray. Once molecules are identified with these screens, the targets of these molecules and their cffects on the checkpoint will be determined. Molecules found with these assays could prove to be useful tools to probe the physiological functions of ATR and the significance of its DNA binding activity. These molecules may also be clinically useful for anti-cancer therapy, particularly when used in combination with chemotherapeutic agents that activate the DNA damage checkpoint or inhibit DNA replication.